Detection of AGO1-associated mRNAs from fly heads. (A) Diagram illustrating the AGO1 IP procedure. For each expressed probe set, an ENR was calculated as a ratio between the expression values in the IP and the INP fractions. The maximum enrichment across samples was used to sort the probe sets in a descending order. This rank order was used in the following calculations. (B) Top 15 enriched mRNAs in the AGO1-IP ranking. “# PicTar” refers to the number of PicTar predicted miRNA-binding sites in the indicated mRNA. (C) Density of PicTar predicted miRNA targets across the AGO-IP enrichment ranking. The inset represents the enrichment P-value for top X ranking genes, with X moved from 50 to 12,847 with increments of 50. (D) Position in the AGO1-IP enrichment ranking of 10 circadian-relevant mRNAs.

Identification of circadian-relevant miRNAs. (A) Determination of miRNAs expressed in tim cells. The plot represents the difference of expression between control (tim-gal4) and the averaged signal between Tim-DroshaIR and Tim-PashaIR flies among the X, second, and third chromosomes of Drosophila (see the Materials and Methods for preparation of the samples). Green arrows indicate the coincidence of a significant peak (P-value < 1 × 10⁻³) with the location of a known miRNA. (B) Close caption of A for three miRNAs: miR-8, bantam, and miR-9a. The signal (in yellow) corresponds to the ratio of signals from Tim-DroshaIR/Tim-PashaIR and control flies. The red arrows indicate the location of the mature miRNA. The genes located around the miRNA sequence are displayed in green and above the yellow signal when their transcription is forward and below when reversed. (C) Detection of Pri-bantam and mature bantam or U6 snRNA by Northern blot. RNA isolated from control (yw) or Tim-DcrIR (tim-gal4; UAS-dcr-1 IR) fly heads were examined by Northern analysis using a specific probe against bantam or U6 snRNA. (D) List of 10 circadian candidates miRNAs (see the text).

Bantam regulates clk 3′ UTR through three evolutionary binding sites. (A) Representation of clk 3′ UTR and conservation between 12 Drosophila species (see the text). Drosophila melanogaster clk 3′ UTR sequence (chr3L:7,756,940–7,757,175; April 2006 genome assembly) and nucleotide conservation between 12 Drosophila genomes were retrieved using the University of California at Santa Cruz genome Web site. The blue line represents the annotated clk 3′ UTR ending at the black arrow (end of clk-sh isoform) (see the text). The red box (B1) indicates the predicted site for the miRNA bantam in the clk 3′ UTR. The green boxes (B2 and B3) refer to the two evolutionary bantam sites located downstream from the annotated clk 3′ end (depicted as the dashed blue line). (B) Effect of modulating bantam levels on 3′ UTR reporters. S2 cells were transfected with a 3′ UTR luciferase reporter (wild-type clk 3′ UTR [left], or mutated bantam sites clk 3′ UTR [B1, B2, and B3] [right]) and either an empty plasmid (pAc; first column), a bantam-expressing plasmid (bantam; second column), or 2′-O-methyl anti-bantam cholesterol-conjugated oligos (ban ASO; third column). Renilla luciferase plasmid was cotransfected for normalization. Two days after transfection, luciferase activity was assayed and data were normalized for each reporter to luciferase levels obtained with the empty plasmid. A representative experiment is shown. For each condition, two experiments with duplicates were performed. Error bars represent standard error of the mean (SEM). (C) Effect of inhibiting the miRNA pathway (by using dsRNA against Ago1) in the levels of clk-lg 3′ UTR reporters. The utilized reporters include a wild-type clk-lg 3′ UTR (left two columns) and a clk-lg 3′ UTR in which the bantam sites have been deleted (last two pairs of columns). (D) Effect of modulating bantam levels on the clk-lg 3′ UTR reporter. S2 cells were transfected with a clk-lg 3′ UTR luciferase reporter and an empty plasmid (pAc; first column), a bantam-expressing plasmid (bantam; second column), or Omethyl anti-bantam cholesterol-conjugated oligos (ban ASO; third column). The ratio of Firefly/Renilla luciferase activity was normalized to the luciferase levels obtained with the empty plasmid for each reporter.

Behavioral characterization of Tim-DcrIR flies. (A) Comparison of circadian locomotor behavior of control flies (tim-gal4; UAS-CYC-VP16; top tracing) and Tim-CYC-VP16-Dcr1IR flies (tim-gal4/UAS-CYC-VP16; UAS-Dcr1 IR bottom tracing) in free-running conditions (DD). The experiment was performed at 29°C. In each case, the behavior is shown in average actograms. (B) Control and Tim-CYC-VP16-Dcr1 IR data sets were smoothed with a low-pass filter set with a cutoff of 12 h.

Clk but not tim 3′ UTR imparts miRNA-mediated regulation to a luciferase reporter in S2 cells. (A) Effects of different 3′ UTRs on the level of expression of a luciferase reporter. Thirty nanograms of pAc-firefly luciferase reporter (containing either the SV40, clk, or tim 3′ UTR) were transfected in S2 cells. Forty-eight hours post-transfection, cell extracts were prepared and firefly luciferase activity was measured. In all cases, cotransfection with pCopia-Renilla Luciferase was performed. For each condition, a normalized Firefly/Renilla Luciferase value was obtained and plotted accordingly. A representative experiment is shown. For each condition, two experiments with duplicates were performed. Error bars represent standard error of the mean (SEM). (B) Similar experiment to the one described in A but the S2 cells were treated for 5 d prior transfection with dsRNA against GFP, Ago1, or ago2. Forty-eight hours post-transfection, cell extracts were prepared, and firefly luciferase activity was measured. The ratios obtained for each dsRNA treatment for the constructs containing the clk and tim 3′ UTRs were normalized to the value in the same condition in the SV40 3′ UTR. The final value was obtained by setting the value with the addition of the control dsRNA to 1.

Behavioral and molecular effects of bantam overexpression in pacemaker cells. (A) Locomotor behavior of control (tim-gal4), Tim-miR14 (tim-gal4; UAS-miR14), and Tim-bantam (tim-gal4; UAS-bantam) flies in DD conditions. The behavior is shown in average actogram (top) and autocorrelation analyses (bottom). The period was calculated for each strain by averaging the periods of individual flies. The error refers to standard error of the mean (SEM). (B) Locomotor behavior of control (UAS-bantam/+) and pdf-bantam (pdf-gal4; UAS-bantam) in DD conditions. The behavior is shown in an average actogram. (C) Luciferase recordings from fly wings of control (tim-luc;tim-gal4) and Tim-bantam (tim-luc;tim-gal4; UAS-bantam) flies using the tim-luciferase reporter. Light timing is indicated by alternating white and gray background areas with white representing the illuminated interval of LD (ZT0–ZT12) and gray representing the dark period (ZT12–ZT24). After 3 d in LD conditions, the assay was conducted in DD. The results are the average of 24 Tim-bantam and 24 control pairs of fly wings. For each data point, the background (defined as the steady-state value for each strain after 7 d in DD) was subtracted, and the resulting value was normalized to the maximum for each strain across the experiment.

The evolutionarily conserved bantam sites are necessary for circadian rhythmicity. (A) Scheme of the clk gene construct used to rescue arrhythmic clk^AR flies (see the text). The three solid arrows indicate the three evolutionarily conserved bantam sites in the clk 3′ UTR. The dotted arrow indicates the fourth potential bantam site in the clk 3′ UTR. (B) Table indicating the percentage of rhythmic flies of the different strains. Flies were entrained for 3 d in LD conditions and then released into DD. Rhythmicity was determined as described in the Materials and Methods. The average RI of rhythmic flies refers to the averaged rhythm index (see the text for details). The differences between flies rescued with control or mutant clk transgenes were assessed by t-test. The obtained P-values are the following: for comparison 1, P < 0.015; for comparison 2, P < 0.0001; for comparison 3, P < 0.015; for comparison 4, P < 0.05; for comparison 5, P < 0.01; for comparison 6, P < 0.05.